Enhanced reproductive success revealed key strategy for persistence of devastated populations in Himalayan food‐deceptive orchid,Dactylorhiza hatagirea

Anthropogenic disturbances adversely affect populations of rare and endemic plants, resulting in reduction of their population size and performance. Among different plant groups, deceptive terrestrial orchids are vulnerable and possess greater extinction risks because of rarity in occurrence. To understand the response of food‐deceptive terrestrial orchids to disturbances, we selected Dactylorhiza hatagirea as our representative species, which is endemic to Himalaya, and studied its natural populations. This species is rare for being habitat specific, pollination limited and threatened in its natural habitats. We tested the hypothesis that disturbances lead to reduction in population size and plant performance of food‐deceptive terrestrial orchids. For assessing the impact of disturbance, two contrasting groups, heavily devastated (HD) and lightly devastated (LD), were identified on the basis of frequency and intensity of disturbance (harvesting of plant for tubers) by interviewing local people, medicinal plant extractors and shepherds. HD sites, in comparison to LD sites, were found to have smaller population sizes, but showed an increase in plant growth traits (plant height, specific leaf area, leaf N and specific shoot length). Similarly, plants at HD sites were found to have invested less in inflorescence (inflorescence size, inflorescence length, inflorescence length fraction and flowers per length), but despite that showed higher reproductive success. This was a clear indication of enhanced performance of its populations driven by disturbances. Our findings suggested that food‐deceptive species in small populations tend to reduce the probability of population extinction and have the capability to recover rapidly if conserved in time.     

Absence of ClC5 in knockout mice leads to glycosuria, impaired renal glucose handling and low proximal tubule GLUT2 protein expression.

Glycosuria is one of the well-documented characteristics in ClC-5 knockout (KO) mice and patients with Dent's disease. However, the underlying pathophysiology of its occurrence is unknown. In this study, we have compared ClC-5 KO mice with age and gender matched wild-type (WT) control mice to investigate if the underlying cause of manifested glycosuria is an impairment of glucose homeostasis and/or an alteration in expression levels of proximal tubule (PT) glucose transporters. We observed that, the blood glucose concentration (n=12, p<0.01) and the fractional excretion of glucose and insulin (n=6, p<0.05) were higher in KO mice. In contrast, the fasting blood glucose levels (n=7) were not significantly different in the two groups. Plasma glucose increased to a greater extent in KO mice (n=7, p<0.05) when challenged by an intraperitoneal injection of glucose. However, no peripheral tissue insulin resistance was observed following an intraperitoneal injection of insulin (n=9) in the KO mice. ELISA analysis demonstrated low plasma insulin concentrations after a 12 hour fasting period and also following glucose injection in KO mice. The total insulin released during a 2 hour period following glucose challenge was significantly lower in KO mice (n=6, p<0.05). By western blot, we observed a significant decrease in GLUT2 protein expression levels in isolated PT ((n=10, p<0.01)) of KO mice. This decrease in protein levels was corroborated by a significant decrease in GLUT2 mRNA levels estimated semi quantitatively by RT-PCR in isolated PT (n=10, p<0.01). No significant changes in mRNA expression levels of SGLT2, SGLT1 and GLUT1, as analyzed by RT-PCR, could be detected in the isolated PT (n=10). Also, we have shown by western blot analysis that expression of megalin is lower in the renal cortex of KO mice when compared to WT mice (n=3, p<0.05). Our results suggest that low plasma insulin concentration together with renal function changes observed in KO mice significantly contribute towards the glucose intolerance and documented glycosuria observed in this animal.     

Detection of an indole oxidizing system in maize leaves

An enzyme activity which brings about a rapid indole disappearance has been detected in cell free extracts of maize ($\text{Zea}\text{mays}$ L.) leaves. The indole utilization by this enzyme system is not dependent on L-serine and pyridoxal phosphate. It does not result in incorporation of (5-³H) indole or (1-¹⁴C) serine into tryptophan. There was no net tryptophan synthesis concomittant with indole disappearance. The enzyme activity is strongly inhibited by dithionite and diethyl-dithiocarbamate. The inhibition by the latter could be specifically removed by Cu²⁺. The activity of dialyzed enzyme could be restored by addition of Cu²⁺ and FAD. The products of indole oxidation were characterized as anthranilic acid and anthranil (2,1-benzisooxazole). The activity of the indole oxidizing system was 2 to 3 times higher in normal maize varieties (Ganga-2 and Ganga-5) than in Opaque-2.     

Structural features in the model of a thermostable and stress-resistant protein, SP1 from aspen

A three dimensional theoretical model of SP1 (stable protein 1), which is resistant to high temperature and biotic-stresses, is presented here. The model was generated by the application of homology modeling technique. The conformational rigidity imparted to the fold by the presence of hydrogen-bonded, C5, C7, C10 and C13 structures in the loop regions, multiple aromatic--aromatic interactions at the protein interior and on the surface, in addition to salt-links and hydrogen-bonds are primarily the major factors, responsible for the increased stability of protein. The putative protein family is characterized by motifs, E-x(0,1)-L-x-[AEGQS] and V-x(2,3)-L-x-[ADEGST] and the active site in the tertiary structure is formed by conserved aromatic and isoleucine clusters.     

Digestive enzyme patterns and evaluation of protease classes in Catla catla (family: Cyprinidae) during early developmental stages

Digestive enzymes of Catla catla were studied during ontogenic development. Specific amylase activity was 0.12+/-0.01 mg maltose mg protein(-1) h(-1) in fish 4 days after hatching (DAH) and reached a maximum on (0.41+/-0.12 mg maltose mg protein(-1) h(-1)) 34 DAH. Total protease activity was minimum (123.2+/-16.5 mU mg protein(-1) min(-1)) on day-8 and reached its highest level (2713+/-147.2 mU mg protein(-1) min(-1)) on day-32. Trypsin activity showed constant increasing trend from day-16 onwards and was maximum on day-34 (118.1+/-7.09 mU mg protein(-1) min(-1)). Highest chymotrypsin activity was found on day-32 (1789.0+/-111.7 mU mg protein(-1) min(-1)). Lipase activity was detected in 4 DAH catla. Lipase activity increased steadily from day-22 onwards. SDS-PAGE of crude enzyme extracts showed that high molecular mass bands (41.8-127.8 kDa) appeared during the early stages followed by low molecular mass bands (17.8-37.2 kDa). The number of protease activity bands in substrate SDS-PAGE increased with age of fish. During ontogenesis of carp, soybean trypsin inhibitor (SBTI), PMSF and TLCK inhibited 75.5+/-1.19% to 92.8+/-0.85%, 53.3+/-9.47% to 90.5+/-2.6% and 39.8+/-3.8% to 84.7+/-1.54% of total protease activity, respectively. There was only 2.58+/-0.66% to 10.21+/-0.09% inhibition of protease activity with EDTA. SBTI and PMSF inhibited 8 and 4 activity bands, respectively. TLCK, a specific trypsin inhibitor, inhibited four trypsin-like enzymes in carp during ontogenesis.     

Methionine limitation results in increased hepatic FAS activity, higher liver 18:1 to 18:0 fatty acid ratio and hepatic TAG accumulation in Atlantic salmon, Salmo salar

The current experiment aimed to study whether interactions with lipid metabolism possibly might explain the relative increased liver weight obtained in fish fed sub-optimal methionine levels. A basal diet based on a blend of plant proteins which is low in methionine (1.6 g Met/16 g N) was compared to a methionine adequate diet (2.2 g Met/16 g N) prepared by adding dl-methionine (2.4 g/kg) to the basal diet in the expense of wheat grain. Fish oil was used as the lipid source. The diets were balanced in all nutrients except methionine. The diets were fed to Atlantic salmon (500 g BW) for a period of 3 months. Feed intake did not differ, rendering the intake of all nutrients except methionine equal. Fish fed the low methionine diet had an increased liver size relative to body weight, indicating fat deposition in the liver. Fish given the sub-optimal methionine diet showed about six times higher fatty acid synthase (FAS) activity as compared to the fish fed the adequate methionine diet, indicating a higher de novo lipogenesis. A significant rise in the liver 18:1 to 18:0 fatty acid ratios also supported storage of lipids over fatty acid oxidation. Indeed, methionine limitation resulted in significantly higher TAG concentrations in the liver. Sub-optimal dietary methionine also resulted in lower hepatic taurine concentrations and the total bile acids concentrations were reduced in faeces and tended to be reduced in plasma. Taken together, our data show that salmon fed sub-optimal methionine levels had increased relative liver weight and developed signs commonly described in the early stage of non-alcoholic fatty liver disease in rodent models (increased FAS activity, changed fatty acid ratios and TAG accumulation).